Stimulation of Calcium/NOS/CK1α Signaling by Cedrol Triggers Eryptosis and Hemolysis in Red Blood Cells

Abstract

Background Cedrol (CRL) is a sesquiterpene alcohol present in the essential oils of coniferous trees including Cupressus and Juniperus genera. CRL has shown potent anticancer activity by virtue of apoptosis. Red blood cells (RBCs), although devoid of mitochondria and nucleus, can undergo hemolysis and eryptosis which contribute to chemotherapy-induced anemia (CIA). In this work, we explored the hemolytic and eryptotic potential of CRL in human RBCs as a safety assessment of the sesquiterpene as an anticancer agent. Methods RBCs from healthy donors were treated with anticancer concentrations of CRL for 24 h at 37°C with varying experimental manipulations. Hemolysis was photometrically assessed by measuring hemoglobin release whereas flow cytometry was employed to detect phosphatidylserine (PS) exposure by annexin-V-FITC, intracellular Ca2+ by Fluo4/AM, cell volume by forward scatter (FSC), and oxidative stress by 2′,7′-dichlorodihy-drofluorescein diacetate (H2DCFDA). Results Significant, concentration-responsive hemo-lysis was noted upon CRL exposure with concomitant K+, LDH, and AST leakage. CRL also significantly increased annexin-V-positive cells and Fluo4 fluores-cence and reduced FSC. Moreover, the cytotoxicity of CRL was significantly ameliorated in the presence of L-NAME, D4476, and PEG 8,000 but was aggravated by urea and sucrose. Conclusion CRL stimulates hemolysis and eryptosis characterized by PS exposure, Ca2+ overload, and cell shrinkage. The hemolytic activity of CRL was mediated through nitric oxide synthase and casein kinase 1α. Blocking either enzyme may attenuate the toxicity of CRL to RBCs and prevent undesirable side effects associated with its anticancer applications.